The actin cytoskelon has been implicated in endocytosis, yet the specific roles for actin are obscure. Moreover, few molecules that link these systems have been identified. The studies proposed here focus on one such linker protein, Hip1R, and on the associated proteins Abp1 and GAK. Each protein is a mouse homologue of a yeast protein discovered and studied in great detail in the Drubin laboratory. The proposed studies provide a unique opportunity to apply to mammalian cells principles learned from studies in yeast so that the specific roles for actin in endocytosis may be elucidated. Hip1R associates intimately with clathrin-coated vesicles and binds directly to clathrin, actin filaments, and PIP2. It is related to yeast Sla2p. Dominant negative approaches and antibody microinjections will be used to test using real time analysis and electron microscopy the in vivo importance of Hiup1R and its domains for endocytosis and for the ultrastructure of the endocytic pathway. A recently developed procedure will be used to reconstitute on lipid bilayers Hip1R with clathrin, actin, and adaptor proteins, testing for roles in clathrin assembly, pit formation, and vesicle fission. Yeast Abp1p functions in endocytosis and activates the Arp2/3 complex. Mouse Abp1 binds to dynamin through its SH3 domain and to actin filaments through its N-terminus, and co-localizes with the Arp2/3 complex in PDGF- treated cells. Biochemical, cell biological, and molecular-genetic approaches will be used to study the functions and interactions of mouse Abp1. Actin Regulating Kinases (ARK) are key regulators of actin organization and endocytosis in budding yeast, and they interact physically with both Sla2p and Abp1p. GAK is a mammalian ARK-like protein kinase that, like Hip1R, associates with clathrin coated vesicles. GAK function in vivo will be elucidated using dominant negative constructs. Moreover, conventional and chemical genetic approaches will identify GAK targets. Effects of GAK phosphorylation on its targets and how GAK is regulated will be determined. In sum, studies proposed here will benefit from a strong foundation of genetic research on the actin cytoskeleton and endocytosis in budding yeast, and will provide novel insights into the roles for actin in endocytosis and the underlying mechanisms and regulatory strategies in mammalian cells.